The present invention relates to a method for producing gradient gel medium membrane for electrophoresis for determining the base sequence of DNA or DNA partially decomposed material or for separating/analyzing protein, particularly protein having a wide range of molecular-weight distribution, such as protein in urine.
Conventionally, in a plane electrophoretic method, high-molecular concentration gradient gel of acrylamide having no self-supporting property has been obtained by forming gel in layers having concentrations different from each other and stacked on one support or between two supports in the electrophoretic direction. Such a gradient gel has been used as a membranous material.
In the method in which gel is formed on a support when it is to be used, however, there has been a disadvantage in that the gel can sometimes be broken or damaged by dropping a material other than a sample onto the gel, when the gel is being formed on the support, when the gel is set in an electrophoretic tank, when the gel is in a state of preservation, or when the gel is added with a sample to be analyzed, and therefore close attention and much skill are required in operation.
In a vertical electrophoretic method in which gel is formed in a mold formed by two glass plates or the like, and electrophoretic analysis is performed while holding the mold vertically, on the other hand, there has been a disadvantage in that it is difficult to make the mold uniform in thickness, and a high degree of skill is required in operation to pour a gel forming solution into a narrow mold before the gel forming solution has gelled.
Particularly, in an operation for determining the base sequence of DNA, it is desirable to form an elongated sheet of gel so that pieces of DNA, as many as possible in number, can be analyzed using one sheet of gel. Difficulties in the production and handling of such an elongated sheet of gel, however, have been encountered. Further, since glass plates are used, there has been a disadvantage in that the glass plates are apt to be broken.
Recently, for industrially producing a gradient gel material for electrophoresis, there has been disclosed a method for producing gradient gel for electrophoresis including the steps of: preparing an aqueous solution or a water dispersion liquid of a mixture of acrylamide monomers and a cross-linking agent; adding a free radial generating material for initiating polymerization of the monomers by the absorption of light irradiation on the solution; forming the solution into the shape of a desired gel product; and adjusting the period of time of light irradiation on the monomer solution so as to change the porosity of the gel when the formed solution is subject to irradiation with light so as to cause polymerization and cross-linking in the monomer solution or the monomer dispersing liquid (see European Pat. No. 0169397A).
There have been further disclosed a method of producing gradient gel for electrophoresis having a concentration gradient of a polymer in the electrophoretic direction including the steps of: supplying two kinds of aqueous solutions differing in concentration from each other, each containing monomers, a cross-linking agent and a polymerization initiator, into a forming device while mixing the aqueous solutions with the mixing ratio therebetween gradually changed; and completing polymerization of the monomers with the cross-linking agent in the forming device; etc. (Japanese Pat. Publication Nos. 61-22903 and 61-39617).
For coating a continuously moving web with a gel forming solution for use for electrophoresis, a general coating head such as shown in FIG. 1 has been used. That is, the electrophoresis gel forming solution used as a coating liquid 12 is supplied in a pressurized state to a cavity 19 in a coating head 18 by a metering pump 22. The cavity 19 is employed as a liquid pool for supplying the coating liquid 12 to a slot 23 communicated with a slot opening 24 and extending over the coating width. The coating liquid 12 enters the slot 23 from the cavity 19, and is applied onto a web 13 on a coating roller 20 from the slot opening 24. In order to stabilize the coating process, a differential pressure chamber 21 is used so as to provide back pressure behind the coating bead.
The former method, however, has problems in that the cost is high because of the required light irradiation equipment and poor productivity, the resolution is low because of increased thickness of the gel, and it is impossible to obtain gradient gel having stability and reproducibility because the reaction initiator causes polymerizing and cross-linking reactions in the presence of light even after completion of production. The latter producing method, on the other hand, has problems in that the productivity is poor because it is a batch-type system, and it is difficult to uniformly branch a gel forming solution for electrophoresis when it is poured into forming devices, so that it is impossible to obtain gradient gel with reproducibility.
Further, products coated using the conventional coating head have problems in that the transverse film thickness is insufficient in uniformity, and in that foreign matter is generated during running over long periods, making it necessary to overhaul and clean the coating head.